PROJECT SUMMARY The limbus contains a small subpopulation of rare limbal stem cells (LSC) that continually repopulates the corneal epithelium. Patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in conjunctivalization of the corneal stroma that triggers neovascularization, chronic inflammation, and ultimately blindness due to an irreversibly opaque cornea. Several approaches have been used to replace LSC by transplanting limbal tissue or ex vivo expanded limbal cells. These procedures have obtained some success, mainly using autologous limbal tissue from patients with unilateral LSCD. However, these treatments remain technically challenging. Moreover, the larger population of patients with bilateral LSCD has no source of autologous LSC and much less success was observed with allogeneic limbal tissue transplants, indicating new approaches are needed for successful treatment of bilateral LSCD patients. Our collaborative research group has now discovered two new sources of purified donor ABCB5+ stem cells for patients with bilateral LSCD (i) allogeneic immunosuppressive ABCB5+ LSC, and (ii) autologous multipotent dermal-skin-derived ABCB5+ DSC (Dermal Stem Cells). We recently discovered that the ABCB5 gene, a new member of the ATP-binding cassette (ABC) superfamily of active transporters, identifies multipotent stem cells in the limbus (LSC) and the skin (DSC) in mice and humans. Importantly, ABCB5 is a cell surface protein and specific monoclonal antibodies developed by our laboratories are capable of prospectively isolating pure ABCB5-positive stem cells from the limbus and skin. Recently published proof-of-principle experiments demonstrated that purified human ABCB5+ (but not ABCB5 negative) LSC were capable of long-term restoration of the corneal epithelium in an immunodeficient mouse model of LSCD, indicating that this purified LSC population has the potential to significantly improve therapy for corneal disease associated with LSCD. The goal of the current grant application is to translate our laboratory research findings into new ABCB5+ stem cell-based therapies to treat unilateral and bilateral LSCD patients. Our overall hypothesis is that ABCB5+ stem cells from the limbus or skin can be isolated and expanded in vitro as a source of stem cells to regenerate the corneal epithelium when transplanted to recipients with either a unilateral or bilateral LSCD. The three Modules of this project will: Validate the regenerative potential of purified in vitro-expanded ABCB5+ LSC (Module 1) and purified in vitro-expanded ABCB5+ DSC (Module 2) for unilateral and bilateral LSCD patients; and (Module 3) create an LSC Biobank for clinical transplantation and conduct studies resulting in FDA IND approval.